Abstract

To develop and evaluate new therapeutic strategies for the treatment of human cancers, well-characterised preclinical model systems are a prerequisite. To this aim, we have established xenotransplantation mouse models and corresponding cell cultures from surgically obtained secondary human liver tumours. Established xenograft tumours were patho- and immunohistologically characterised, and expression levels of cancer-relevant genes were quantified in paired original and xenograft tumours and the derivative cell cultures applying RT-PCR-based array technology. Most of the characteristic morphological and immunohistochemical features of the original tumours were shown to be maintained. No differences were found concerning expression of genes involved in cell cycle regulation and oncogenesis. Interestingly, cytokine and matrix metalloproteinase encoding genes appeared to be expressed differentially. Thus, the established models are closely reflecting pathohistological and molecular characteristics of the selected human tumours and may therefore provide useful tools for preclinical analyses of new antitumour strategies in vivo.

1. Introduction

The liver is a common site of
distant metastasis originating from different neoplasms including
gastrointestinal (pancreatic, stomach, colorectal), lung and breast cancers.
Also primary liver tumours such as cholangiocellular carcinomas (CCC), cancers
of the bile ducts [1], may disseminate into the liver. Surgical
resection still is the most promising therapy of secondary liver tumours, however,
only a minority of patients are candidates for resection, and no adjuvant
treatment has been demonstrated to be effective in increasing the survival rate
following radical surgery [2, 3]. For unresectable disease, several
treatments have been tested in the clinical setting; however, none of them can
be currently considered a standard approach. This also applies to systemic
chemotherapy, although newer regimens appear to at least improve median
survival [4]. Locoregional therapies such as
hepatic intra-arterial chemotherapy and isolated hepatic perfusion may be
offered to patients with unresectable liver metastases in the absence of
extrahepatic disease; however, the efficacy of these treatments is still being
determined. Both systemic and locoregional chemotherapy might be useful in the
neoadjuvant setting to increase the resectability of liver metastases initially
not amenable to surgical resection.

Due to its poor prognosis and unsatisfying
treatment options, suitable animal models for secondary liver cancer are
required as a prerequisite for studying factors involved in the pathogenesis of
the disease as well as for the development and evaluation of new anticancer
therapies. Various approaches include the use of transgenic or knockout mice
[5, 6] or mouse models, in which tumour
formation is induced chemically [7]. Albeit tumours develop in all of
these mouse models, tumour formation and progression in mice greatly differ from that in man [8, 9] due to physiological differences
between the species and differences in cellular and molecular events
contributing to cancer development. Tumour models established with primary human
tumour tissue may overcome some of these limitations. To this aim, immune compromised
animals, such as severe combined immunodeficient (SCID) mice, are grafted either
subcutaneously or orthotopically with cultured cells [10, 11] or tissue derived from human tumour
material [12–15] providing convenient models for
evaluation of distinct anticancer strategies, especially those targeting tumour
growth. Although discussions are ongoing arguing that the orthotopic
transplantation model closer resembles the situation in the patient,
subcutaneous xenografts still remain the standard for cancer drug screening in
the pharmaceutical industry. In both cases, only detailed knowledge about the
transplanted tumour cells will facilitate correct interpretation of gained
results.

Thus, in the present
study liver metastases derived from various human adenocarcinomas were used to establish
subcutaneous xenograft tumours in SCID/beige mice. Extensive histological analyses
were performed to demonstrate that the transplants widely reflect the
characteristics of the parental lesion. In addition, gene expression profiling
by means of RT-PCR-based microarrays revealed that expression of cancer-related
genes appeared to be similar in corresponding original and xenograft tumours as
well as in derived cell cultures. Therefore, we conclude that the established
tumour models and cell cultures may represent valuable tools for the
development and analysis of new treatments targeting secondary liver tumours.

Tumour
pieces either obtained from primary (AKH23, KFJ18) or xenografted tumours
(AKH10, KFJ6, KFJ9, KFJ10) were processed as described above, and obtained
single cell suspensions were transferred into cell culture flasks (Sarstedt)
containing culture medium. Established
cell cultures were characterised by immunocytochemistry using antibodies
reacting with human and mouse major histocompatibility complex (MHC) class I
antigens. Briefly, cells were incubated with a R-phycoerythrin—conjugated mouse
anti-human leukocyte antigen (HLA)-A,B,C (BD Pharmingen, Schwechat, Austria) or a fluorescein isothiocyanate
(FITC)—conjugated mouse
antimouse H-2Dd monoclonal antibody (Becton Dickinson, Heidelberg, Germany) for one hour at 4°C in the dark. Cells
were washed twice, resuspended in PBS, and subjected to FACS analysis
(FACScalibur, Becton Dickinson). In addition, cells were stained with an
antibody directed against a human epithelial-specific antigen (ESA; Serotec, Düsseldorf, Germany) followed by detection with FITC-conjugated
polyclonal rabbit anti-mouse immunoglobulin (DakoCytomation, Glostrup, Denmark). After
characterisation, cells usually with passage numbers 5–10 were frozen in
liquid nitrogen for long-term storage. On demand cells were thawed and expanded
for further in vitro analysis
or retransplantation into immunodeficient mice. Therefore, cells were injected subcutaneously into SCID/beige
mice as described above. In addition to tumour growth in vivo, anchorage
independent growth of recultivated tumour-derived cells was analysed by colony
formation in a standard soft agar assay [17].

2.4. Histopathological Analysis and Immunohistochemistry

Xenograft
tumours after the first or second passage in mice were excised and fixed in 4%
buffered formalin (pH 7.0, Sigma-Aldrich) and embedded in paraffin (Histo-Comp,
Sanova, Wien, Austria)
using automatic embedding equipment (Tissue Tek, Miles Scientific, Inc., Ill, USA). Three μm thick
sections of primary and xenograft tumours were routinely stained with
haematoxylin and eosin and microscopically analysed. To characterise primary
tumours and corresponding xenografts by immunohistochemistry, the following
primary antibodies were used: rabbit polyclonal antibody specific for
carcinoembryonic antigen (CEA, CD66e Ab-2, neat, Labvision Neomarkers, Cheshire, UK), mouse
monoclonal antibodies specific for cytokeratin 8/18 (CK8/18 Labvision
Neomarkers, 1:100 diluted in PBS) and cytokeratin 20 (CK20, DakoCytomation;
1:50 diluted in PBS). For detection of CK8/18, sections were digested with 0.1%
protease (Sigma-Aldrich) in PBS for 15 minutes. CK20 was detected after pretreatment
with 0.1% proteinase K (Sigma-Aldrich) in PBS. Sections were then incubated
with 1.5% goat serum (DakoCytomation) for 30 minutes followed by overnight
incubation with the primary antibody at 4°C. Detection was performed using the Vectastain
ABC-AP kit (Vector Laboratories, England, UK) with New Fuchsin (DakoCytomation) as a substrate
followed by counterstaining with Mayer’s haemalum (VWR International GmbH, Dresden, Germany). Sections
were covered with Aquatex (Merck,
Darmstadt, Germany) and
examined by light microscopy (Zeiss
Axiovert 200 M, Carl Zeiss GmbH, Oberkochen, Germany).

2.5. RNA Extraction, Reverse Transcription, and Quantitative RT-PCR

RNA was extracted from
trypsinised cells or frozen and pulverised tumour samples according to the
RNeasy Mini Kit protocol (Qiagen, Wien,
Austria) and treated afterwards
with Turbo DNase (Ambion, Tex,
USA) according to the manufacturer’s instructions. Subsequently, 150 ng
of total RNA were reverse transcribed using the iScript cDNA synthesis Kit (Bio-Rad
Laboratories, Calif, USA). 50 μL of cDNA template
(105 ng total input RNA) were amplified using a master mix containing 1x reaction buffer B (Solis Biodyne, Tartu, Estonia), 5 mM MgCl2, 0.2 mM of
each dNTP (Applied Biosystems, Calif, USA), 300 nM ROX reference dye (Invitrogen, Lofer, Germany) and 1 unit
of hot start Firepol polymerase (Solis Biodyne, Tartu, Estonia) on TaqMan low density arrays
(Applied Biosystems) using the ABI PRISM 7900HT sequence detection system
(Applied Biosystems). The respective human-specific real-time PCR primers and
probes are listed in Table 1. According to data base comparisons (Applied
Biosystems), these sequences are not supposed to cross-react with mouse. Cycling
conditions were as follows: 2 minutes at 50°C, 10 minutes at 94.5 followed by
40 cycles of 30 seconds at 97°C and 1 minute at 59.7°C. Ct values were
determined using the passive reference dye and manual baseline and threshold
settings in the SDS 2.2 software (Applied Biosystems). Assays with Ct values
above 33 were excluded from analysis due to variations or inappropriate
amplification in duplicate wells. Tumour-derived RNA was tested in duplicates
on three different plates, and their mean values were calculated for further
analysis. A calibrator sample consisting of a universal reference RNA isolated
from 10 different human cancer cell lines (Stratagene, Calif, USA) served as an internal standard
for comparison of different assays. Differences in gene expression levels of each
tumour sample were first normalised to the calibrator sample followed by
calculation of differences between original and xenograft tumours according to the method [18]. Normalisation of real-time RT-PCR
data was performed using the geometric mean (normalisation factor) of the included
endogenous reference genes GUSB (ß-glucuronidase), ACTB (ß-actin) and rRNA18S
(18S ribosomal RNA) within the macros-based program qBase
(http://medgen.ugent.be/qbase).

Table 1: Sequences amplified on TaqMan low density
arrays.

2.6. Statistical Analysis

To
identify genes expressed differentially in all xenografts and parental tumours
analysed, a Wilcoxon paired-samples test was performed (SPSS for Windows Vs. 11.5). Statistical
significance was defined as P < .05. For the analysis of individual
original/xenograft tumour pairs, we considered genes to be differentially
expressed showing a minimum of 2.5-fold difference between xenograft and
original tumours.

3. Results

Human secondary
liver tumour tissue was obtained from patients at the time of surgery or
resection of the neoplasm. In total, tumour samples from 17 patients including liver
metastases of colorectal carcinomas (), intrahepatic cholangiocellular
carcinomas () as well as a metastasis of a pancreatic carcinoma were
collected. The tumour tissue was digested with collagenase to obtain single
cell suspensions which were injected subcutaneously into SCID/beige mice.
Finally, injection of single cells prepared from 10 different samples consisting
of liver metastases
originating from colorectal (), cholangiocellular (), and a pancreatic adenocarcinoma
resulted in tumour formation. The main characteristics of the original
xenografted tumour samples are summarised in Table 2.

Table 2: Relevant characteristics of original human tumour samples.

3.2. Histopathological Features of Original Human Tumours Are Conserved in Corresponding Xenografts

In order to compare original and
xenograft tumours morphologically, sections were stained with haematoxylin/eosin
and examined by light microscopy. Representatively for colorectal liver
metastases, sections of the original tumour KFJ6 and its derived xenograft are
shown (see Figures 1(a) and 1(b)). Both original as well as the xenograft tumours revealed irregular
tubular structures typical for colon adenocarcinomas. In most of the established xenograft tumours, large areas of necrosis were observed
(data not shown). The tumour AKH10 is depicted as an example of an intrahepatic
cholangiocellular carcinoma (see Figures 1(c) and 1(d)). Pathohistologically,
both xenograft and the parental tumour can be described as a moderately
differentiated adenocarcinoma with comparable simple tubular to glandular structures.
Examination of the liver metastasis AKH23 which had originated from a pancreatic
adenocarcinoma revealed a solid undifferentiated large cell carcinoma (see Figure 1(e)). Tumour cells exhibited anaplastic nuclei and varying amounts of
eosinophilic, particular foamy cytoplasm. Consistently, subcutaneous implantation
of cells derived from tumour AKH23 led to formation of a poorly differentiated
fast growing anaplastic carcinoma (see Figure 1(f)).

To further
characterise the established xenograft tumours and their corresponding original
counterparts, immunohistological stainings for detection of CEA were performed.
CEA is a glycoprotein expressed in adenocarcinomas of the intestinal tract and
in other tumours of epithelial origin such as lung adenocarcinoma, pancreatic
adenocarcinoma, and cholangiocellular carcinomas (CCCs) [19]. Additionally, tumours were stained with
antibodies specific for CK8/18, which is expressed in simple and glandular
epithelia, and CK20, which is primarily expressed in colon adenocarcinomas.
Pancreatic tumours and CCCs may also express CK20 [20]. As summarised in Table 3, immunohistological
analyses revealed similar staining patterns within original and xenograft
tumour samples with regard to expression of CEA, CK8/18, and CK20. Positive
staining exclusively detected in distinct original tumour samples was due to
reactions with normal liver cells no more present in the xenograft tumours. Representative
analyses of original tumours and their corresponding xenografts are shown in Figures
2–4. Immunohistological comparison of the original and xenograft tumour KFJ6 in
both samples revealed expression of CEA in the cytoplasm and membranes of luminal
cells (see Figures 2(a) and 2(b)). Additionally, expression of CK8/18 (see Figures
2(c) and 2(d)) and CK20 (see Figures 2(e) and 2(f)) was detected. AKH10
original and xenograft tumours (see Figure 3) both reacted with antibodies
specific for CEA (see Figures 3(a) and 3(b)) and CK8/18 (see Figures 3(c) and
3(d)) but did not show expression of CK20 (see Figures 3(e) and 3(f)). The original
pancreatic adenocarcinoma-derived liver metastasis AKH23 (see Figure 4) revealed
single cells expressing CEA (see Figure 4(a)), whereas the corresponding
xenograft tumour appeared negative for CEA expression. A robust staining in
both samples was obtained when expression of CK8/18 (see Figures 4(c) and 4(d))
was analysed. In contrast, neither original nor xenograft tumour-derived
sections revealed expression of CK20 (see Figures 4(e) and 4(f)). Based on
these findings, we conclude that the investigated human tumours retained their typical
morphological and histological characteristics after xenotransplantation into
mice.

In order to compare the established xenograft
tumour models with the respective original tumour counterparts on a molecular
basis, gene expression analyses were performed. For this purpose, relative
expression levels of a number of cancer-relevant genes (see Table 1) were
determined in the respective corresponding tumour samples using TaqMan low density
expression arrays. Interassay specific differences were first normalised to an
arbitrarily chosen calibrator (reference RNA), and then the ratio of gene expression
levels in an original tumour versus the corresponding xenograft tumour was
determined. Genes were considered to be differentially expressed when a
2.5-fold minimal difference between original and xenograft tumour samples was obtained.
Table 4 summarises data acquired for a representative selection of
different original tumours in comparison to their respective xenografts.
Interestingly, genes encoding cell cycle regulators and proto-oncogenes, such as
Bcl-2, cyclin D1, CDC25B, cyclin-dependent kinase inhibitor 1B, Erb-b2, K-ras,
Met and Myc as well as epidermal growth factor receptor (EGFR) and a ß-catenin
encoding gene (CTNNB1), showed comparable expression levels in all the investigated
original and xenograft tumours. Expression of the proto-oncogene Wnt-1 was
neither detected in original nor in xenograft tumour tissue. In contrast, genes
encoding cytokines such as interleukin 8 (IL-8) and 6 (IL-6), its receptor
IL6-R, cyclooxygenase (Cox)-2, vascular endothelial growth factor (VEGF)-C as
well as matrix metalloproteinase (MMP) 11 appeared to be differentially
expressed in some of the analysed samples (see Table 4). In particular, IL-6,
Cox-2, and VEGF-C expression was nearly exclusively detected in original tumour
samples. Expression of IL-6 receptor (IL-6R) was either found to be equal in
original and xenograft tumours or significantly increased (4- to 18-fold) in
some of the original tumours (AKH10, KFJ18, KFJ21). Similarly, IL-8 appeared to
be 12- to 100-fold higher expressed in original tumour samples compared to the
corresponding xenograft tissue. Analysis of MMP-11 expression revealed a 4- to 22-fold difference between original and
xenograft tumours. Although
few more differences were encountered concerning expression of serpin and VEGF
(AKH23), statistical analysis of results obtained for all investigated original
and xenograft tumour samples revealed significant differences exclusively for
the expression of IL-8 (P = .017) and MMP-11 (P = .018).

Table 4: Relative differences in gene expression levels (n-fold) of
original tumour samples compared to the corresponding xenograft
tumour. Indicated
values represent the mean of three measurements including the calculated
standard deviation. Ratios were calculated after normalisation of individual
RNA amounts to a standard reference RNA. Values indicating differences higher
than 2.5-fold are printed in bold. Gene symbols correspond with Table 1. n.d.: not determinable, Ct values obtained
with cDNA derived either from the xenograft (#) or from both tumour samples were
below threshold (>39).

Finally, gene expression
levels of original and xenograft tumour samples exemplarily were compared to those
of their derived cell cultures (see Table 5). Immunocytochemical
characterisation of established cell cultures confirmed their human and
epithelial origin, respectively (data not shown). Again, the most striking
differences in expression levels were observed for IL6-R and MMP encoding
genes. IL6R-expression levels were about 5-fold decreased in tumour-derived cell
cultures compared to the corresponding tissue. Demonstrative differences in
MMP-1 expression were observed for AKH23-derived cells, which showed a >300-fold
higher amount of mRNA compared to the parental tumour. In contrast, MMP-11 (10-fold)
and VEGF (4-fold) expression levels were found to be higher in AKH23 original
tumour tissue.

4. Discussion

Tumour mouse models as well as
tumour-derived cell lines are a prerequisite for the development and evaluation
of new and existing tumour therapies. Although a number of xenograft models have been published for colorectal
carcinomas and pancreatic adenocarcinomas in most cases, these were established
from cultured cell lines available for example from ATCC. In these examples, it
is not clear how long-term cultivation of these (mostly poorly characterised)
cells affects tumour formation and biology. Therefore, we decided to establish
xenografts directly from patient tumours and subsequently analyse both tissues
in detail to demonstrate that the generated model closely reflects the original
malignancy. In the present study, we report the
establishment and detailed characterisation of human xenograft tumour models
derived from secondary liver cancer, that is, tumour metastases originating
from colorectal, cholangiocellular, and pancreatic cancers. Xenografts were
established directly from tumour biopsies omitting culturing of isolated cells,
which may cause development of tumours that do not share the characteristics of
the respective original due to the selection and expansion of specific cell
clones. The applied method of enzymatic digestion of whole tumour samples followed
by injection of a mixture of tumour and stromal cells was shown to overcome
this obstacle. With respect to xenografts derived from colorectal carcinomas,
the applied method resulted in a take rate of 60% and 50%, respectively, when
cholangiocellular carcinoma-derived cells were injected. Retrospective analysis
of xenograft tumour growth with clinical data of the respective patient did not
reveal any significant correlation. Instead, the condition of the primary
tumour sample, for example, the presence of large necrotic areas appeared to be
critical.

Pathohistological examination of the
established xenografts and comparison to their respective original tumours
demonstrated that the typical morphology of the tumours was retained after
xenotransplantation. Moreover, immunohistological analyses showed that each of
the established xenograft tumours retained the typical tumour-specific antigen
profile observed in the original tumour sample. Cell cultures established
either from original or xenograft tumour tissues were shown to be of epithelial origin and
not contaminated with murine cells (data not shown). Although the respective
tumour transplants could be passaged in mice for extended periods (up to 30
times) without major changes in growth behaviour and morphology (data not
shown), a cryoconservation protocol was established facilitating storage of
samples at early passages to avoid development of histopathological alterations
over time. Retransplantation experiments with tumour samples frozen for
different time spans (3, 6, and 12 months) revealed an average take rate of 70% to 100% in both SCID/beige and nude mice.

Molecular
characterisation based on quantitative gene expression analyses using human
specific primers and probes revealed that in most of the corresponding original
and xenograft tumour samples expression of oncogenes and genes involved in cell
cycle regulation appeared not to be affected by the xenografting process. Major
differences within original and xenograft tumour samples as well as their
derived cell cultures were detected regarding genes encoding cytokines (IL-8,
IL-6) and matrix metalloproteinases (MMP-1, MMP-11). This finding can be
explained by the fact that these molecules are rather expressed by inflammatory
cells (monocytes, neutrophils), stromal fibroblasts, and endothelial cells than
by the tumour cells themselves. A high level IL-8 expression, however, was also
reported in cultured colon carcinoma cells, where it was associated with the
metastatic behaviour of these cells [21]. Consistently, we have shown IL-8 expression in cultured xenograft-derived
colon carcinoma cells (e.g., KFJ10), and their metastatic potential was
demonstrated by colony formation in soft agar assays (data not shown).

Matrix
metalloproteinases (MMPs) are a family of extracellular matrix degrading
enzymes, which have their physiological role in tissue remodelling processes
such as embryonic development or wound healing [22]. In cancer, MMPs are described to
be involved in tumour invasion, metastasis, and angiogenesis [23, 24]. MMP-1, also known as interstitial
collagenase, is expressed in a wide variety of cells such as stromal
fibroblasts, endothelial cells, macrophages, and epithelial cells [25]. Either equal expression levels
were found in original and xenograft tumours or expression was exclusively
detected in original tumours. A weak or lacking MMP-1 expression in some of the
xenograft tumours could not be linked to an individual tumour type. Original
tumours representing liver metastases showed higher MMP-1 levels, reflecting the
potential of tumour cells to invade and metastasise from their original site to
distant organs [26]. Accordingly, AKH23 primary tumour-derived
cells exhibiting a markedly high MMP-1 expression level demonstrated a very aggressive
growth behaviour when injected into immunodeficient mice. Injection of 5 × 106 cells in this case resulted in growth of tumours of up to 1000 mm3 within 35 days whereas in average xenografted cells took 60 to 80 days to reach
this tumour volume (data not shown). MMP-11 in comparison to MMP-1 is described
to be specifically expressed in stromal fibroblasts surrounding tumour cells [27]. Thus, the determined reduced
expression level of MMP-11 in xenograft tumours most probably is due to the absence
of human stroma cells in the murine environment. Interestingly, expression of
Cox-2 (PTGS2) and VEGF-C, both known to regulate angiogenesis and
lymphangiogenesis, was detected in original tumour samples but, in contrast to
VEGF-A, was beyond detection limits in most of the xenograft tissues. Recently,
it has been described that these two genes are coexpressed in human colorectal
carcinoma cells and can be significantly associated with lymph node metastasis
and prognosis [28]. Further investigation of the
mechanisms of down regulation of expression of lymphangiogenesis inducing
factors in xenografted tumours may give insight into metastatic progression of CRC.

5. Conclusion

The developed carefully characterised
human xenograft tumours derived from secondary liver tumours share assertive
characteristics with their respective original human counterparts. In addition,
the established cell cultures offer the possibility to evaluate new therapeutic
strategies in vitro before
their use in vivo in the
corresponding tumour mouse models. These valuable tools might be used for the
development and preclinical evaluation of new therapeutic drugs as well as of
alternative methods such as expression targeted retroviral vectors [29] or liver specific therapeutic nanoparticles [30] generated for an application in cancer gene therapy.

Acknowledgments

The authors thank Bettina
Grasl-Kraupp and Hannes Zwickl, Institute of Cancer Research, Medical
University Vienna, and Stefan Stättner, Department of Surgery,
Kaiser-Franz-Josef-Spital Vienna, for providing primary human liver tumour
tissue. They also thank Michaela Wendl and Marielle König-Schuster for taking
care of the animals. In addition, they acknowledge the excellent technical
assistance of Doris Rosenfellner and the support and technical advice of Ingrid
Walter, both at the University of Veterinary Medicine, Institute of Histology and Embryology. The
authors also appreciate the help of Irene Sommerfeld-Stur, Institute of Animal
Breeding and Genetics, in statistical analysis of the presented data. This work
was funded by the Austrian Genome Research Program GEN-AU
GZ200.058/6-VI/2/2002. The work of M. Stürzl was funded by a grant provided from the Interdisciplinary Center for Clinical Research (IZKF) of the University of Erlangen-Nürnberg.